Comparison of the Time-Dependent Changes in Immediate Early Gene Labeling and Spine Density Following Abstinence From Contingent or Non-contingent Chocolate Pellet Delivery

Abstract

Rationale: Incubation of craving is a phenomenon whereby responding for cues associated with a reward increases over extended periods of abstinence. Both contingent and non-contingent behavioral designs have been used to study the incubation of craving phenomenon with differing results. The present study directly compares behavioral and neural changes following contingent or non-contingent administration of chocolate flavored pellets.Objective: The current study examined whether an incubation of craving response would be observed at the behavioral and neural levels following delays of abstinence from chocolate pellets in a contingent or non-contingent reinforcement design.Methods: Rats were trained for 10 days to bar press for chocolate pellets (contingent) or received chocolate pellets in a non-contingent design (classical conditioning). Groups were then subjected to abstinence from the reward for 24 h, 7, 14 or 28 days at which point they were tested for responding for reward associated cues. Following the test, brains from all rats were processed and assessed for c-Fos and FosB labeling as well as dendritic spine density in the nucleus accumbens (NAc).Results: Behavioral measures during the test (lever presses, food hopper entries and locomotor activity) revealed similar behavioral outcomes across all delays indicating the lack of an incubation of craving response on both the contingent and non-contingent designs. Overall, labeling of c-Fos in the NAc was lower for the non-contingent group compared to the operant-trained and food restricted control. Compared to the operant-trained and non-trained control groups, a significantly reduced FosB labeling was noted in the NAc of the classically conditioned groups across all abstinence periods. Spine density in the NAc was elevated in both the classically and operant conditioned compared to the food-restricted, non-trained controls.Conclusions: Chocolate pellet reward did not result in incubation of craving but did produce behavioral learning that was associated with increased spine density. This suggests that chocolate pellet administration results in long-term structural and functional changes that are present for at least 28 days following abstinence. Contingent and non-contingent administration resulted in differential immediate early gene labeling in the NAc, but the functional significance of this has yet to be elucidated.

Evaluating Time-Dependent Changes in Food Seeking Following Periods of Forced Abstinence and Associated Changes in the Nucleus Accumbens

“…The chocolate pellet group also displayed a higher total spine density compared to both milk chocolate and control rats. We have previously shown that consumption of chocolate flavored pellets in an operant task resulted in a prolonged increase in total dendritic spine density in the NAcSh compared to food restricted controls (Noye Tuplin et al, 2018). The total spine density in the chocolate pellet group remained steady over time, whereas in the control group, total spine density dropped between the 14 day and 28-day abstinence periods.…”

“…FosB and c-Fos immunohistochemistry followed similar staining procedures as previously reported (Noye Tuplin, et al, 2018). Two sections with the NAc from each rat were chosen for FosB and two other sections were chosen for c-Fos immunohistochemical staining (bregma 2.7-3.2).…”

“…The brain hemispheres not processed for c-Fos or FosB were prepared for Golgi impregnation as previously described (Noye Tuplin, et al, 2018). For each of the three groups (control, chocolate flavored pellets, milk chocolate), nine secondary/tertiary dendrites on MSNs within the NAcSh per rat, with a minimum of 3 rats per abstinence period, were analyzed.…”

“…In human populations, chocolate is considered one of the most craved foods in North America (Nasser et al, 2011). We have previously studied incubation of craving for chocolate flavored pellets in a self-administration model, where rats acquire robust responding for pellets by day 5 of acquisition (Davis-MacNevin et al, 2013;Noye Tuplin et al, 2018;Tuplin et al, 2015). Chocolate flavored pellets differ in macronutrient composition and lack vital chocolate components (cocoa) compared to chocolate consumed by the human population.…”

“…To determine if the rewarding effects of chocolate flavored pellets are similar to real chocolate, the chocolate pellet group was compared to a milk chocolate food group. We have previously shown that rats acquire food-seeking behavior in response to discrete environmental cues previously paired with noncontingent chocolate pellet delivery (Noye Tuplin et al, 2018) but the previous model would not allow for the dispensing of milk chocolate chips. The present study utilized an unbiased, non-contingent, CPP procedure to explore the incubation of craving for chocolate flavored pellets or milk chocolate chips and to determine any differences between the two foods in the ability to produce time-dependent changes in CPP.…”